Today, biology is in many ways the science of proteins and nucleic acids. Nucleic acids are found in all living matter. For each species or host, unique sequences exist providing for the genotype and phenotype of that particular host. Thus, one can use the presence of a particular sequence as indicative of the particular strain or species. In many instances, a number of strains will share a common sequence as distinct from other strains or species, so that one can not only detect a particular strain but, if desired, can detect subspecies, species or genera. In addition, one can distinguish between RNA or DNA so as to determine whether a particular gene is being expressed, the existence of one or more alleles, the level of expression, and the like. Where cells, such as B-cells and T-cells, are involved with genomic rearrangements, one can detect the presence or absence of such rearrangements by employing probes. Thus, the detection of particular nucleic acid sequences is a powerful tool in the diagnosis of disease states, the presence of sets or subsets of cells, the particular strain or species of a pathogen, such as a bacterium, protista, or virus, or the like.
The detection and isolation of sequences is also important in the field of molecular biology. Thus, the use of probes allows for detection of a variety of sequences of interest, including structural genes, regulatory sequences, introns, exons, leader sequences, both translated and untranslated, and the like.
There is also substantial interest in detecting sequences in genetic engineering. Monitoring levels of transcription, detecting the integrity of constructs, monitoring levels of mutation, resection, mapping, or the like provide opportunities for nucleic acid screening and detection.
In many instances, the sequence of interest may be present as only a very small fraction of the total amount of nucleic acid, and/or in very small amount, e.g. attomole levels. Furthermore, the sequence of interest may be accompanied by a number of sequences having substantial homology to the sequence of interest. Thus, relatively high stringencies may be required to ensure the absence of unwanted hetero-duplexing, which may further limit the available concentration of the sequence of interest.
Additionally, the same or similar sequences may appear on nucleic acid fragments of different size and the appearance of a sequence on a particular size fragment may be correlated to the presence of a particular phenotype.
There is also interest in developing analytical systems which can be automated, so as to minimize the time and energy required from technicians, as well as minimizing errors which may result from manual manipulation. In many systems the sample is labeled to allow for detection of the sequence. The labeling can be time consuming and limited as to the nature of the label as in nick translation with radioactive nucleotide triphosphates. In other situations, the particular nature of the label may be limited, as when using terminal deoxytransferase. Other techniques result in random substitution. There is therefore an interest in providing for rapid convenient controlled labeling of sample nucleic acids, where the labeled moiety may be commercially available and require little, if any, technical skills in being used to label the sample.